EP1311666A2 - Gene 13 (psgen 13) a progression supprimee et ses utilisations - Google Patents

Gene 13 (psgen 13) a progression supprimee et ses utilisations

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Publication number
EP1311666A2
EP1311666A2 EP01964487A EP01964487A EP1311666A2 EP 1311666 A2 EP1311666 A2 EP 1311666A2 EP 01964487 A EP01964487 A EP 01964487A EP 01964487 A EP01964487 A EP 01964487A EP 1311666 A2 EP1311666 A2 EP 1311666A2
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European Patent Office
Prior art keywords
cell
nucleic acid
protein
sequence
seq
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EP01964487A
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German (de)
English (en)
Inventor
Paul B. Fisher
Dong-Chul Kang
Zao-Zhong Su
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Columbia University in the City of New York
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Columbia University in the City of New York
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Publication of EP1311666A2 publication Critical patent/EP1311666A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a gene which is expressed at lower levels in cells as they progress toward a malignant phenotype, and hence is referred to as Progression Suppressed Gene 13 ("PSGen 13").
  • PSGen 13 Progression Suppressed Gene 13
  • the invention is based, at least in part, on the discovery of this novel gene and its encoded protein, and on the discovery that introducing PSGenl3 into a malignant cell inhibits cancer cell growth and inhibits expression control elements of genes associated with malignancy and blood vessel formation.
  • DDRT-PCR is a powerful methodology in which a vast number of mRNA species (>20,000, if no redundancy occurs) can be analyzed with only a small quantity of RNA ( approx. 5 ⁇ g) (Id.).
  • DDRT-PCR is often the method of choice when the RNA source is limiting, such as tissue biopsies.
  • a direct advantage of DDRT-PCR is the ability to identify and isolate both up- and down-regulated differentially expressed genes in the same reaction.
  • the DDRT-PCR technique permits the display of multiple samples in the same gel, which is useful in defining specific diagnostic alterations in RNA species and for temporally analyzing gene expression changes.
  • DDRT-PCR technique is not problem-free (Debouck, 1995, Curr. Opin. Biotechnol. 6: 597-599). Difficulties encountered when using standard DDRT-PCR include a high incidence of false positives and redundant gene identification, poor reproducibility, biased gene display, and lack of functional information about the cloned cDNA. Furthermore, poor separation can mask differentially expressed genes of low abundance under the intense signals generated by highly expressed genes. The generation of false positives and redundancy can be highly problematic, resulting in an inordinate expenditure of resources to confirm appropriate differential expression and uniqueness of the isolated cDNAs.
  • cDNAs must be isolated from the gels in pure form (contamination of bands with multiple sequences complicates clone identification), reamplified, placed in an appropriate cloning vector, analyzed for authentic differential expression, and, finally, sequenced.
  • Subtractive hybridization in which hybridization between tester and driver is followed by selective removal of common gene products, enriches for unique gene products in the tester cDNA population and reduces the abundance of common cDNAs (Sagerstr ⁇ m, et al., 1997, Annu. Rev. Biochem. 66: 751-783).
  • a subtracted cDNA library can be analyzed to identify and clone differentially expressed genes by randomly picking colonies or by differential screening (Rangnekar et al., 1992, J. Biol. Chem. 267: 6240-6248; Wong et al, 1997, Semin. Immunol. 9: 7-16; Maser and Calvet, 1995, Semin. Nephrol. 15: 29-42).
  • DDRT-PCR performed with subtracted RNA or cDNA samples should provide a powerful strategy to clone up- and down-regulated gene products.
  • This approach should combine the benefits of both techniques, resulting in the enrichment of unique sequences and a reduction or elimination of common sequences.
  • This scheme also should result in a consistent reduction in band complexity on a display gel, thereby permitting a clearer separation of cDNAs, resulting in fewer false positive reactions.
  • rare gene products that are masked by strong common gene products should be displayed by using subtraction hybridization in combination with DDRT-PCR.
  • Kang et al. (1998, Proc. Natl. Acad. Sci. U.S.A. 95:13788-13793) used a reciprocal subtraction differential RNA display (“RSDD") approach that efficiently and consistently reduces the complexity of DDRT-PCR and resulted in the identification and cloning of genes displaying anticipated differential expression.
  • the model used for RSDD was an adeno virus-transformed rat embryo cell line, El 1, that acquires an aggressive onco genie progression phenotype when injected into athymic nude mice and reestablished in cell culture (Ell -NMT) (Su and Fisher, 1997, Proc. Natl. Acad. Sci. U.S.A.
  • PEGen genes progression- elevated gene
  • PSGen genes suppression-suppressed gene
  • the present invention relates to the cloning and characterization of the complete rat and human PSGen 13 cDNAs, and provides their nucleic acid and encoded amino acid sequences, and moreover demonstrates the suppressive effects of increased levels of PSGen 13 on the malignant phenotype.
  • the invention provides for isolated nucleic acids encoding Progression Suppressed Gene 13 (PS Gen 13) proteins, vectors comprising said nucleic acids, isolated PSGen 13 proteins, and methods of using such molecules to prevent the growth of cancer cells and/or new blood vessels and accordingly to treat patients suffering from cancer. It is based, at least in part, on the cloning and characterization of the complete cDNAs encoding rat and human PSGen 13, and on the discovery that increased levels of PSGen 13 expression can suppress the transformed phenotype and inhibit the activities of promoter elements associated with cancer progression and angiogenesis.
  • the present invention provides a method for inhibiting growth of a cancer cell which comprises contacting the cancer cell with a nucleic acid encoding a PSGen 13 protein, a PSGen 13 protein or a PSGen 13 activator compound in a sufficient amount so as to inhibit growth of the cancer cell.
  • the invention also provides a method for treating cancer in a subject which comprises contacting a cell of the subject with a nucleic acid encoding a PSGen 13 protein in a sufficient amount so as to cause the cell to express the PSGen 13 protein, thereby treating cancer in the subject.
  • a DNA "coding sequence” or a “nucleotide sequence encoding" a particular protein is a DNA sequence which is transcribed and translated into a polypeptide in vivo or in vitro when placed under the control of appropriate regulatory sequences.
  • the boundaries of the coding sequence are determined by a start codon at the 5'- (amino) terminus and a translation stop codon at the 3' - (carboxy) terminus.
  • a coding sequence can include, but is not limited to, procaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) sources, viral RNA or DNA, and even synthetic nucleotide sequences.
  • a transcription termination sequence will usually be located 3' to the coding sequence.
  • DNA "control sequences” refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, untranslated regions (“UTRs"), including 5'-UTRs and 3 '-UTRs, which collectively provide for the transcription and translation of a coding sequence in a host cell.
  • a control sequence "directs the transcription" of a coding sequence in a cell when an RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
  • hancer element is a nucleotide sequence that increases the rate of transcription of a therapeutic gene or genes of interest but does not have promoter activity.
  • the term "gene” refers to a nucleic acid directly or indirectly encoding a product.
  • cDNA encoding a PSGen 13 protein would be considered a "PSGen 13 gene", as would genomic DNA from which a PSGen 13-encoding mRNA could be transcribed.
  • a "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature.
  • heterologous coding sequence is a construct where the coding sequence itself (e.g., the coding sequence of PSGen 13 mutant or fragment of nucleic acid encoding PSGen 13) is not found in nature (e.g., synthetic sequences having codons different from the native gene).
  • a chimeric sequence comprising a heterologous structural gene and a gene encoding a PSGen 13 or a portion of such gene, linked to a tissue specific promoter, whether derived from the same or a different gene, will be considered heterologous since such chimeric constructs are not normally found in nature. Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA, as used herein.
  • nucleic acid molecule includes both DNA and RNA and, unless otherwise specified, includes both double-stranded and single-stranded nucleic acids. Also included are hybrids such as DNA-RNA hybrids. Reference to a nucleic acid sequence can also include modified bases as long as the modification does not significantly interfere either with binding of a ligand such as a protein by the nucleic acid or Watson-Crick base pairing, unless such interference is intended or desirable.
  • operably linked refers to an arrangement of nucleotide sequence elements wherein the components so described are configured so as to perform their usual function.
  • control sequences operably linked to a coding sequence are capable of effecting the expressing of the coding sequence.
  • the control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • Two DNA or polypeptide sequences are "substantially homologous" when at least about 80% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule.
  • substantially homologous also refers to sequences showing at least about 80%, preferably at least about 90% and more preferably at least about 95% identity to the specified DNA or polypeptide sequence.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for the particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Ausubel et al., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc. New York.
  • therapeutic gene means DNA encoding an amino acid sequence corresponding to a functional protein capable of exerting a therapeutic (e.g., growth inhibitory, differentiating, and/or apoptosis inducing) effect on cancer cells or having a regulatory effect on the expression of a function in cells.
  • a cell has been "transformed” by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane or, in the case of bacteria, the cell wall.
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In procaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid.
  • a stably transformed cell is generally one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication, or one which includes stably maintained extrachromosomal plasmids. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA.
  • FIGURE 1 Nucleotide and predicted amino acid sequence of the rat
  • PSGen 13 gene (designated rPSGen 13), SEQ ID NOS: 1 and 2, respectively. Starting ATG of the open reading frame and the stop codon are circled and the poly(A) signal is underlined .
  • FIGURE 2 Nucleotide and predicted amino acid sequence of the human PSGen 13 gene (designated HuPSGen 13), SEQ ID NOS : 3 and 4, respectively. The starting ATG of the open reading frame and stop codon are circled and the poly(A) signal is underlined.
  • FIGURE 3 Nucleotide sequence comparison between the rat PSGen 13 ; SEQ ID NO: 1) and human (HuPSGen 13; SEQ ID NO: 3) cDNAs. The start and stop codons of the rat PSGen 13 and human HuPSGen 13 genes are in bold-face type.
  • FIGURE 4 Amino acid sequence comparison between the rat PSGen 13 and human HuPSGen 13 proteins (SEQ ID NOS: 2 and 4, respectively). Conservative substitutions are indicated by a ":” and semi-conservative substitutions are indicated by a ".”.
  • FIGURE 5 Differential expression of rPSGen 13 identified by RSDD and reverse Northern blotting in a large panel of rodent cells displaying differences in transformation progression.
  • RNA was obtained from cell types represented in the Northern blot in lanes, left to right, as follows: unprogressed El 1 cells ("Ell (-)"); progressed El 1-NMT (+) cells; CREF x El 1-NMT FI (-) unprogressed cells (where "Cell Type A x Cell Type B” designates a somatic cell hybrid between a Type A cell and a Type B cell); CREF x El 1-NMT F2 (-) unprogressed cells; CREF x El 1-NMT Rl (+) progressed cells; CREF x El 1-NMT R2 (+) progressed cells; Ell x El 1-NMT A6 (-) unprogressed cells; Ell x El 1-NMT A6TD (+) nude mouse tumor-derived progressed cells; El 1 x El 1-NMT 3b (-) unprogressed cells; El 1 x El 1-NMT Ila (+) progressed cells; El 1-NMT AZA Bl (-) un
  • RNAs Equal loading of RNAs is demonstrated by ethidium bromide (EtBr) staining. Data is from Kang et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:13788-13793.
  • FIGURE 6 rPSGen 13 suppresses anchorage independent growth in El 1-NMT cells.
  • Agar cloning efficiencies of the indicated cell types were determined as described previously (Fisher et al., 1979, Cancer Res. 39:3051-3057; Fisher et al., 1979, Nature 281:591-594; Fisher et al, 1979, Cell 18:695-705).
  • Cell types include, El 1, El 1-NMT and PSGen 13 transfected El 1-NMT clones, designed as NMT- PSG13 cl 3, 5, 6, 7, 8, 9, 10, 11 and 12. 'Triplicate samples varied by ⁇ 10% and replicate assays varied by ⁇ 15%.
  • FIGURE 7 Northern blotting analysis of rat PSGen 13 and GAPDH expression in El 1, El 1-NMT and NMT-PSG13 clones. Fifteen micrograms of cellular RNA isolated from the indicated cell types were electrophoresed, transferred to nylon membranes and hybridized with a rat PSGen 13 cDNA and then stripped and probed with GAPDH as previously described (Su et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:9125-9130; Su et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96:15115-15120).
  • FIGURE 8 Rat PSGen 13 inhibits anchorage independent growth in DU-145 human prostate carcinoma cells.
  • Cell types include, DU-145, DU-145 vector transformed clone (DU- 145/Vec) and rat PSGen 13 transfected DU-145 clones, designed as DU-PSG13 cl 11, 12, 13, 14, 15 and 17. Triplicate samples varied by ⁇ 10% and replicate assays varied by ⁇ 12%.
  • FIGURE 9 Full length PEG-3 promoter-luciferase activity in El 1 , El 1-NMT and NMT-PSG13 clones. Different cell types were co-transfected with 5 ⁇ g of the full length PEG-Prom and 1 ⁇ g of a pSV- ⁇ -galactosidase plasmid and luciferase activity was determined as described in Materials and Methods 48 hr later. The results are standardized by ⁇ -galactosidase activity and represent the average of 3 independent experiments that varied by ⁇ 15%.
  • FIGURE 10 VEGF promoter-luciferase activity in El 1, El 1-NMT and NMT-PSG13 clones.
  • VEGF-Prom-luciferase Different cell types were co-transfected with 5 ⁇ g of the VEGF-Prom-luciferase (Su et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96:15115- 15120) and 1 ⁇ g of a pSV- ⁇ -galactosidase plasmid and luciferase activity was determined as described in Materials and Methods 48 hr later. The results are standardized by ⁇ -galactosidase activity and represent the average of 3 independent experiments that varied by ⁇ 15%.
  • FIGURE 11 HuPSGen 13 selectively suppressed monolayer colony formation in Ha-r ⁇ _? transformed rat embryo fibroblast cells.
  • FIGURE 12 HuPSGen 13 suppressed the transformed phenotype of MCF-7 human breast carcinoma cells, as demonstrated by a decrease in cloning efficiency in agar.
  • PSGen 13 nucleic acids for clarity of presentation, and not by way of limitation, the detailed description of the invention is divided into the following subsections: (i) PSGen 13 nucleic acids; (ii) PSGen 13 proteins; (iii) antibodies to PSGen 13 proteins; and (iv) uses of PSGenl3.
  • the present invention provides for PSGen 13 nucleic acids, including PSGen genes and related molecules.
  • PSGen 13 refers to a PSGen 13 molecule without regard to species.
  • the present invention provides for a rat PSGen 13 ("rPSGen 13") gene having a sequence as set forth in FIGURE 1 and SEQ ID NO: 1 or SEQ ID NO:5 and as deposited with the American Type Culture Collection (“ATCC”), an organization having an address at 10801 University Boulevard., Manassas, VA 20110-2209 and assigned Accession Number PTA-2414, and substantially homologous molecules.
  • ATCC American Type Culture Collection
  • the deposit is a rPSGen 13 c DNA insert in a pcDNA3.1(+) plasmid vector, within the EcoRI- Xho I cloning site.
  • the insert is about 0.8 kb in length.
  • the sense strand promoter of the plasmid is T7.
  • the plasmid carries resistance genes to ampicillin and neomycin.
  • the insert origin is EST clone ATCC # 2005777 (see below).
  • the rat tissue used to isolate the cDNA was adrenal gland tissue.
  • the present invention provides for a human PSGen 13 ("HuPSGen 13") gene having a sequence as set forth in FIGURE 2 and SEQ ID NO:3 or SEQ ID NO:6, and as deposited with the ATCC and assigned Accession Number PTA-2413, and substantially homologous molecules.
  • the deposit is an insert within a plasmid vector, pT7T3-Pac. It is within the EcoRI- Not I cloning site. The insert is about 0.83 kb in length.
  • the plasmid carries a resistance gene to ampicillin.
  • the insert origin is EST clone ATCC #2525262 (see below).
  • the human tissue used to isolate the DNA was human kidney tissue.
  • the present invention further provides for nucleic acids which would hybridize with the rPSGen 13 gene and/or the HuPSGen 13 gene under stringent hybridization conditions, such as e.g., hybridization in 0.5 M NaHPO 4 , 7 percent sodium dodecyl sulfate (“SDS”), 1 mM ethylenediamine tetraacetic acid (“EDTA”) at 65°C, and washing in O.lx SSC/0.1 percent SDS at 68°C (Ausubel et al., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, hie, and John Wiley & Sons, Inc. New York, at p. 2.10.3).
  • stringent hybridization conditions such as e.g., hybridization in 0.5 M NaHPO 4 , 7 percent sodium dodecyl sulfate (“SDS”), 1 mM ethylenediamine tetraacetic acid (“EDTA”) at 65°C, and washing in O.lx SSC/0.1
  • the present invention encompasses PSGen 13 genes of other species, including a PSGen 13 gene of a non- human primate, a mouse, or a cow, as well as nucleic acid probes and antisense molecules (which could be used, for example, to support a transformed phenotype).
  • the present invention also provides for nucleic acids encoding the amino acid sequences set forth for the rPSGen 13 and HuPSGen 13 proteins in FIGURES 1 and 2, and as set forth in SEQ ID NOS: 2 and 4 , and for nucleic acids that hybridize to such PSGen 13 protein-encoding nucleic acids under stringent conditions.
  • the nucleic acid sequences set forth in FIGURES 1 and 2 may be used to identify primer molecules which may be used to obtain a PSGen 13 gene by amplification, using standard techniques.
  • the present invention provides for a PSGen 13 gene, such as the rPSGen 13 gene or the HuPSGen 13 gene, in an expressible form.
  • An "expressible form” is one which contains the necessary elements and control sequences for transcription and/or translation.
  • a PSGen 13 gene may be operatively linked to a suitable promoter element (which may be a PSGen 13 promoter or a heterologous promoter), and may comprise an enhancer element, transcription initiation and termination sites, nucleic acid encoding a nuclear localization sequence, ribosome binding sites, polyadenylation sites, mRNA stabilizing sequences, etc..
  • a PSGen 13 gene such as the rPSGen 13 gene or the HuPSGen 13 gene, in a form expressible in eucaryotic cells.
  • the PSGen 13 gene may be operatively linked, for example, to a promoter element active in eucaryotic cells.
  • Suitable promoters may include the cytomegalovirus immediate early promoter, the Rous sarcoma virus long terminal repeat promoter, the human elongation factor l ⁇ promoter, the human ubiquitin c promoter, etc.. It may be desirable, in certain embodiments of the invention, to use an inducible promoter.
  • inducible promoters include the murine mammary tumor virus promoter (inducible with dexamethasone); commercially available tetracycline-responsive or ecdysone-inducible promoters, etc..
  • the promoter may be selectively active in cancer cells, such as the prostate specific antigen gene promoter (O'Keefe et al., 2000, Prostate 45:149-157), the kallikrein 2 gene promoter (Xie et al., 2001, Human Gene Ther. 12:549-561), the human alpha-fetoprotein gene promoter (Ido et al., 1995, Cancer Res. 55:3105-3109), the c-erbB-2 gene promoter (Takakuwa et al., 1997, Jpn. J. Cancer Res.
  • the prostate specific antigen gene promoter O'Keefe et al., 2000, Prostate 45:149-157
  • the kallikrein 2 gene promoter Xie et al., 2001, Human Gene Ther. 12:549-561
  • the human alpha-fetoprotein gene promoter Ido et al., 1995, Cancer Res. 55:3105-3109
  • the human carcinoembryonic antigen gene promoter (Lan et al., 1996, Gastroenterol. 111:1241-1251), the gastrin-releasing peptide gene promoter (Inase et al., 2000, Int. J. Cancer 85:716-719).
  • the human telomerase reverse transcriptase gene promoter (Pan and Koenman, 1999, Med. Hypotheses 53:130-135), the hexokinase II gene promoter (Katabi et al., 1999, Human Gene Ther. 10:155-164), the L-plastin gene promoter (Peng et al., 2001,
  • Cancer Res. 61 :4405-4413 the neuron-specific enolase gene promoter (Tanaka et al, 2001, Anticancer Res. 21:291-294), the midkine gene promoter (Adachi et al., 2000, Cancer Res. 60:4305-4310), the human mucin gene MUC1 promoter (Stackhouse et al, 1999, Cancer Gene Ther. 6:209-219), and the human mucin gene MUC4 promoter (Genbank Accession No. AF241535), which is particularly active in pancreatic cancer cells (Perrais et al., 2001, published on June 19, 2001 by J Biol. Chem., "JBC Papers in Press” as Manuscript Ml 04204200).
  • expressed sequence tag (“EST") clones containing, unbeknownst to the initial depositors, rPSGen 13 and HuPSGen 13 amino acid sequence encoding sequences, were deposited with the ATCC prior to the filing date ofU.S.S.N. 09/648,310 and had been assigned Accession Numbers 2005777 and 2525262, respectively. These clones were used in the characterization and cloning of the complete rPSGen 13 and HuPSGen 13 genes in the present invention (see Section 6, below). However, at the time the ESTs were deposited the complete genes were not known, and neither the identity nor the function of the protein encoded by the ESTs was known.
  • a PSGen 13 nucleic acid such as a rPSGen 13 gene or a HuPSGen 13 gene operatively linked to a promoter element operative in a eucaryotic cell, may be comprised, e.g. together with other expression related elements, gene(s) associated with antibiotic resistance, etc., in a vector molecule.
  • suitable vector molecules include but are not limited to include virus-based vectors and non- virus based DNA or RNA delivery systems.
  • virus-based gene transfer vectors include, but are not limited to, those derived from retroviruses, for example Moloney murine leukemia- virus based vectors such as LX, LNSX, LNCX or LXSN (Miller and Rosman, 1989, Biotechniques 7:980-989); lentiviruses, for example human immunodeficiency virus (“HIV”), feline leukemia virus (“FIV”) or equine infectious anemia virus (“EIAV”)-based vectors (Case et al., 1999, Proc. Natl. Acad. Sci. U.S.A.
  • retroviruses for example Moloney murine leukemia- virus based vectors such as LX, LNSX, LNCX or LXSN (Miller and Rosman, 1989, Biotechniques 7:980-989)
  • lentiviruses for example human immunodeficiency virus (“HIV”), feline leukemia virus (“FIV”) or equine infectious anemia virus
  • Ad5/CMV-based El-deleted vectors for example Ad5/CMV-based El-deleted vectors (Li et al., 1993, Human Gene Ther. 4:403-409); adeno-associated viruses, for example pSub201-based AAV2-derived vectors (Walsh et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:7257-7261); herpes simplex viruses, for example vectors based on HSV-1 (Geller and Freese, 1990, Proc. Natl. Acad. Sci. U.S.A.
  • baculoviruses for example AcMNPV-based vectors (Boyce and Bucher, 1996, Proc. Natl. Acad. Sci. U.S.A. 93:2348-2352); SV40, for example SVluc (Strayer and Milano, 1996,Gene Ther. 3:581-587); Epstein-Barr viruses, for example EBV-based replicon vectors (Hambor et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:4010-4014); alphaviruses, for example Semliki Forest virus- or Smdbis virus-based vectors (Polo et al., 1999, Proc.
  • vaccinia viruses for example modified vaccinia virus (MVA)-based vectors (Sutter and Moss, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10847-10851) or any other class of viruses that can efficiently transduce human tumor cells and that can accommodate the nucleic acid sequences required for therapeutic efficacy. It may be desirable, for certain embodiments, to utilize a shuttle vector.
  • MVA modified vaccinia virus
  • the present invention provides for compositions comprising an above- described PSGen 13 nucleic acid in a suitable carrier.
  • the present invention provides for a therapeutic amount of a PSGen 13 nucleic acid, for example as comprised in expressible form in a vector, in a suitable pharmaceutical carrier.
  • the present invention also provides for a procaryotic or eucaryotic host cell containing a PSGen 13 nucleic acid, as set forth above, for example as comprised in a vector.
  • the host cell may be a procaryotic or eucaryotic cell, including but not limited to a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.
  • the host cell may be a malignant or a non-malignant cell, hi specific non-limiting embodiments of the invention, the host cell may be a human tumor cell, including, for example, a nasopharyngeal tumor cell, a thyroid tumor cell, a central nervous system tumor cell (e.g., a neuroblastoma, astrocytoma, or glioblastoma multiforme cell), a melanoma cell, an epithelial tumor cell, a non-epithelial tumor cell, a blood tumor cell, a leukemia cell, a lymphoma cell, a neuroblastoma cell, a cervical cancer cell, a breast cancer cell, a lung cancer cell, a prostate cancer cell, a colon cancer cell, a hepatic carcinoma cell, a urogenital cancer cell, an ovarian cancer cell, a testicular carcinoma cell, an osteosarcoma cell, a chondrosarcoma cell, a gastric cancer cell, or
  • PSGEN 13 PROTEINS The present invention provides for PSGen 13 proteins, i particular embodiments, the invention provides for a rPSGen 13 protein as depicted in FIGURE 1 and having a sequence as set forth in SEQ ID NO:2, and substantially homologous proteins. In other particular embodiments, the invention provides for a HuPSGen 13 protein as depicted in FIGURE 2 and having a sequence as set forth in SEQ ID NO:4, and substantially homologous proteins.
  • the present invention provides for a PSGen 13 protein encoded by a PSGen 13 nucleic acid as described above, and/or which cross-reacts with an antibody directed toward rPSGen 13 protein and/or HuPSGen 13 protein, as described below. Accordingly, in addition to human and rat PSGen 13 proteins, the present invention also provides for bovine, mouse protein, and non- human primate PSGen 13 proteins.
  • a PSGen 13 protein may be prepared from a natural source, may be chemically synthesized, or may be produced by recombinant DNA techniques.
  • a PSGen 13 protein may be produced by expressing a PSGen 13 protein comprised in a suitable expression vector.
  • a PSGen 13 protein may be expressed using standard techniques in a eucaryotic or a procaryotic expression system. Where a eucaryotic expression system is used, nucleic acid encoding a PSGen 13 protein, preferably comprised in a vector in expressible form, may be introduced into a eucaryotic cell by any standard technique, including transfection, transduction, electroporation, bioballistics, microinjection, etc..
  • the present invention provides for compositions comprising an above- described PSGen 13 protein in a suitable carrier.
  • the present invention provides for a therapeutic amount of a PSGen 13 protein in a suitable pharmaceutical carrier.
  • the invention provides for an antibody which binds specifically to a PSGen 13 protein described herein.
  • the antibody binds specifically to a rPSGen 13 protein having a sequence as depicted in FIGURE 1 and as set forth in SEQ ID NO:2.
  • the antibody binds to a HuPSGen 13 protein having a sequence as depicted in FIGURE 2 and as set forth in SEQ ID NO:4.
  • Such antibody may, in certain instances, cross-react with several PSGen 13 proteins from different species.
  • the antibody may be, for example but not by way of limitation, a human antibody, a murine antibody, a non-human primate antibody, a bovine antibody, a sheep antibody, a goat antibody, or a rat antibody.
  • the antibody may be a murine monoclonal antibody, a humanized monoclonal antibody, a human monoclonal antibody, a humanized primate monoclonal antibody, or a humanized rat monoclonal antibody.
  • a PSGen 13 protein may be used as an immunogen to generate antibodies.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and a Fab expression library.
  • antibodies which recognize rPSGen 13 or HuPSGen 13 are produced.
  • polyclonal antibodies which specifically bind to a PSGen 13 protein.
  • various host animals can be immunized by injection with the native PSGen 13 protein, or a synthetic version, or derivative (e.g., fragment) thereof, including but not limited to rabbits, mice, rats, goats, etc.
  • adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete or incomplete) adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum for preparation of monoclonal antibodies directed toward a PSGen 13 protein, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • monoclonal antibodies can be produced in germ-free animals utilizing recent technology PCT/US90/02545).
  • human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). Further, according to the invention, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A.
  • Antibody fragments which contain the idiotype of the molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 , fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 , fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • the invention provides for a composition which comprises an antibody described herein and a carrier, h particular embodiments of the invention, the composition is a pharmaceutical composition.
  • An antibody according to the invention may be used, for example, in the purification of a PSGen 13 protein or in a method of diagnosis to detect or measure the amount of PSGen 13 protein present, where the amount of PSGen 13 protein would be decreased in a cell as it progresses toward the transformed phenotype.
  • the present invention relates to the use of PSGen 13 to suppress the transformed phenotype of a malignant cell
  • indices of the transformed phenotype include but are not limited to cell proliferation (growth), morphology, lack of contact inhibition, the increased expression of transformation associated genes, the decreased expression of differentiation-specific genes, capability of anchorage-independent growth, lack of onset of senescence or apoptosis, tendency to form tumors and metastasize, etc.
  • the effect of PSGen 13 may be mediated by introducing a PSGen 13 nucleic acid into a malignant cell, in expressible form, and/or by introducing a PSGen 13 protein or a PSGen 13 activator substance (see below).
  • the invention provides for a method for inhibiting growth of a cancer cell which comprises contacting the cancer cell with a nucleic acid encoding a PSGen 13 protein, a PSGen 13 protein, or a PSGen 13 activator substance.
  • a PSGen 13 activator substance increases the level of PSGen 13 present in the cell, for example a substance which induces transcription of the PSGen 13 gene by promoter activation, or which increases the half-life of PSGen 13 protein or mRNA.
  • the cancer cell in particular embodiments, is a human cancer cell, hi specific, non- limiting embodiments, the invention provides for a method for inhibiting growth of a human cancer cell comprising exposing said cell to an effective amount of a rPSGen 13 or HuPSGen 13 nucleic acid (in expressible form) or a rPSGen 13 or HuPSGen 13protein.
  • the human cancer cell may be a nasopharyngeal tumor cell, a thyroid tumor cell, a central nervous system tumor cell (e.g., a neuroblastoma, astrocytoma, or glioblastoma multiforme cell), a melanoma cell, an epithelial tumor cell, a non-epithelial tumor cell, a blood tumor cell, a leukemia cell, a lymphoma cell, a neuroblastoma cell, a cervical cancer cell, a breast cancer cell, a lung cancer cell, a prostate cancer cell, a colon cancer cell, a hepatic carcinoma cell, a urogenital cancer cell, an ovarian cancer cell, a testicular carcinoma cell, an osteosarcoma cell, a chondro sarcoma cell, a gastric cancer cell, or a pancreatic cancer cell.
  • a central nervous system tumor cell e.g., a neuroblastoma, astrocytoma,
  • the invention provides for a method for treating a subject suffering from a cancer, comprising administering, to the subject, a therapeutically effective amount of a nucleic acid encoding a PSGen 13 protein, a PSGen 13 protein, or a PSGen 13 activator substance.
  • the subject in specific non- limiting embodiments, is a human, hi particular non-limiting embodiments, the invention provides for a method for treating a human subject suffering from a cancer comprising administering to said subject a therapeutically effective amount of a rPSGen 13 or HuPSGen 13 nucleic acid (in expressible form) or rPSGen 13 or HuPSGen 13 protein.
  • the cancer to be treated may be a nasopharyngeal tumor, a thyroid tumor, a central nervous system tumor (e.g., a neuroblastoma, astrocytoma, or glioblastoma multiforme), melanoma, a vascular tumor, a blood vessel tumor (e.g., a hemangioma, a hemangiosarcoma), an epithelial tumor, a non- epithelial tumor, a blood tumor, a leukemia, a lymphoma, a neuroblastoma, a cervical cancer, a breast cancer, a lung cancer, a prostate cancer, a colon cancer, a hepatic carcinoma, a urogenital cancer, an ovarian cancer, a testicular carcinoma, an osteosarcoma, a chondrosarcoma, a gastric cancer, or a pancreatic cancer.
  • a central nervous system tumor e.g., a neuroblasto
  • PSGen 13 nucleic acids may be introduced by any method known in the art which may utilize, for example, but not by way of limitation, vector mediated entry (e.g., infection), liposomes (e.g. DC-cholesterol liposomes, cationic liposomes, liposomes containing Sendai virus coat protein), imidazolium lipids (see, for example, United States Patent No. 6,245,520 by Wang et al, issued June 12, 2001), cationic lipids (see, for example, United States Patent No. 6,235,310 by Wang et al., issued May 22, 2001), lipofection, asialoglycoprotein poly(L)lysine complexes, and microbubbles (see, for example, United States Patent No.
  • vector mediated entry e.g., infection
  • liposomes e.g. DC-cholesterol liposomes, cationic liposomes, liposomes containing Sendai virus coat protein
  • imidazolium lipids see
  • a PSGen 13 protein may be introduced into a cell by any method known in the art, including methods which utilize formulations such as liposomes, microspheres, or vehicles that facilitate pinocytosis or phagocytosis.
  • the subject may be administered a therapeutically effective amount of a PSGen 13 gene or protein by any suitable route, including intra-tumor instillation, intravenous, intraarterial, intrathecal, intramuscular, intradermal, subcutaneous, etc..
  • a therapeutically effective amount of these agents produces one or more of the following results: a decrease in tumor mass, a decrease in cancer cell number, a decrease in serum tumor marker, a decrease in tumor metastasis, a decrease in vascularization, a decrease in perfusion, a decreased rate of tumor growth, improved clinical symptoms, and/or increased patient survival.
  • the cancer may be first treated surgically to de-bulk the tumor mass, if appropriate.
  • IDENTIFYING POTENTIAL TARGET CELLS It may be desirable to evaluate whether a particular cancer may be a suitable target for the methods of the present invention. Such an evaluation may be made, for example, by introducing a PSGen 13 gene (in expressible form) or protein into a test cancer cell and determining whether the transformed phenotype of that cell is suppressed, for example, but not by way of limitation, by testing the proliferative capabilities of the cell and/or its ability to form colonies in soft agar.
  • the methods of the invention may further comprise determining whether PSGen 13 can decrease PEG3 expression, for example by suppressing its promoter activity. Such activity may be monitored, in specific embodiments, using a PEG3 promoter/reporter gene construct, such as, but not limited to, the PEG3-Prom/ luciferase gene reporter system exemplified herein.
  • a PEG3 promoter/reporter gene construct such as, but not limited to, the PEG3-Prom/ luciferase gene reporter system exemplified herein.
  • the present invention provides for a method for identifying putative PSGen 13 therapeutic targets by determining whether a cancer cell exhibits an increased level of VEGF.
  • the methods of the invention may further comprise determining whether PSGen 13 can decrease VEGF expression, for example by suppressing its promoter activity.
  • Such activity may be monitored, in specific embodiments, using a VEGF promoter/reporter gene construct, such as, but not limited to, the VEGF-Prom/ luciferase gene reporter system exemplified herein.
  • a VEGF promoter/reporter gene construct such as, but not limited to, the VEGF-Prom/ luciferase gene reporter system exemplified herein.
  • tumors particularly associated with angiogenesis such as vascular tumors such as melanoma or hemangiomas, may be suitable targets for PSGen 13- mediated treatments.
  • HuPSGen 13 has been mapped to the chromosomal region 6q23.2-6q23.3.
  • a cancer cell bearing a deletion in this region may be particularly sensitive to the transformation suppressive effects of HuPSGen 13.
  • the present invention provides for a method for identifying a human cancer cell target for treatment with HuPSGen 13 comprising detecting a deletion in chromosomal region 6q23.2-6q23.3.
  • the invention provides for a method for inhibiting angiogenesis associated with tumor growth in a subject which comprises administering to the subject a pharmaceutical composition comprising a nucleic acid encoding a PSGen 13 protein, a PSGen 13 protein, or a PSGen 13 activator compound in a sufficient amount so as to inhibit angiogenesis associated with tumor growth in the subject.
  • the subject is a human
  • the method of treatment comprises administering a therapeutically effective amount of rPSGen 13 or HuPSGen 13 nucleic acid, in expressible form, or of rPSGen 13 or HuPSgen 13 protein, hi non-limiting embodiments, it may be desirable to administer a PSGen 13 nucleic acid or protein together with another molecule which has anti-angiogenic activity, such as angiostatin or thalidomide.
  • the present invention further provides for diagnostic methods which utilize the chromosomal location of HuPSGen 13, 6q23.2-6q23.3.
  • the invention provides for a method of identifying an individual with an increased risk of developing a cancer, comprising detecting, in the individual, a deletion in chromosomal region 6q23.2-6q23.3.
  • the increased risk refers to the development of a cancer selected from the list including pancreatic cancer, papillary serous carcinoma of the peritoneum, hepatocellular carcinoma, large B cell lymphoma, prostate cancer, breast cancer, gastric cancer, and B cell non-Hodgkins lymphoma.
  • the invention provides for a method of detecting progression of a cancer in a subject (i.e., the development of a more malignant phenotype) comprising detecting, in the individual, a deletion in chromosomal region 6q23.2-6q23.3.
  • the progression occurs in a cancer selected from the list including pancreatic cancer, papillary serous carcinoma of the peritoneum, hepatocellular carcinoma, large B cell lymphoma, prostate cancer, breast cancer, gastric cancer, and B cell non-Hodgkins lymphoma.
  • Deletions may be detected using any method known in the art, including but not limited to restriction fragment length polymorphism analysis.
  • PROGRESSION SUPPRESSED GENE 13 (PSGen 13) INHIBITS THE TRANSFORMED STATE IN RODENT AND HUMAN CANCER CELLS
  • a full-length rodent PSGen 13 gene was generated, placed in an expression vector and stably transfected into a progressed rodent transformed cell line, El 1-NMT, and the DU-145 human prostate carcinoma cell line.
  • El 1-NMT progressed rodent transformed cell line
  • DU-145 human prostate carcinoma cell line A series of random single cell clones were isolated and evaluated for expression of the transformed state as documented by their ability to grow in an anchorage independent manner.
  • El 1-NMT is a subclone of Ell cells derived from a nude mouse tumor induced by the El 1 cell line (Babiss et al., 1985, Science 228:1099-1101).
  • R12 is a Ha-ras oncogene transformed El 1 clone (Duigou et al., 1989, NY Acad Sci. 567:302-306).
  • FI and F2 are suppressed somatic cell hybrids with a flat morphology that were formed between El 1-NMT and CREF cells (Duigou et al., 1990, Mol. Cell. Biol. 10:2027-2034).
  • Rl and R2 are progressed somatic cell hybrids with a round morphology that were created by fusing El 1-NMT and CREF cells (Id.).
  • El 1-HPV E6/E7 is a clone of El 1 cells transformed with E6/E7 gene region of human papilloma virus type 18 (Su et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:9125-9130).
  • El 1 x El 1-NMT A6 and 3b are independent somatic cell hybrid clones formed between El 1 and El 1-NMT cells that do not display the progression phenotype (Reddy et al., 1993, in Chromosome and Genetic Analysis, Methods in Molecular Genetics, Adolph, ed., Academic Press, Inc., Orlando, FL pp. 68-102). El 1 X El 1 -NMT cells that do not display the progression phenotype (Id.).
  • El 1 X El 1-NMT A6TD is a progressed somatic cell hybrid formed by isolating a tumor induced in a nude mouse by the El 1 X El 1-NMT A6 somatic cell hybrid (Su et al., 1997, Proc. Natl.
  • El 1 X El 1-NMT Ila is a somatic cell hybrid formed between El 1 and El 1-NMT that exhibits the progression phenotype (Reddy et al., 1993, in Chromosome and Genetic Analysis, Methods in Molecular Genetics, Adolph, ed., Academic Press, Inc., Orlando, FL pp. 68-102).
  • El 1-NMT Aza Bl and Aza Cl are independent clones of El 1-NMT cells treated with 5- azacytidine and displaying suppression in the progression phenotype (Su et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:9125-9130; Reddy et al., 1993, in Chromosome and Genetic Analysis, Methods in Molecular Genetics, Adolph, ed., Academic Press, Inc., Orlando, FL pp. 68-102).
  • CREF is a specific immortal non-transformed and non- tumorigenic clone of Fischer rat embryo fibroblast cells (Fisher et al., 1982, Proc. Natl. Acad. Sci. U.S.A.
  • DU-145 is a hormone refractive human prostate carcinoma cell line (Jiang et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:9160- 9165). All cultures were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% FBS (DMEM-5) at 37°C in a humidified 5% CO 2 / 95% air incubator. Anchorage independent growth assays were performed as described previously by seeding variable numbers of cells in 0.4% noble agar containing medium on a base layer of 0.8% noble agar containing medium (Kang et al., 1998, Proc. Natl. Acad. Sci. U.S.A.
  • NMT-PSG13 clones cl 3, 5, 6, 7, 8, 9, 10, 11 and 12
  • DU- PSG13 clones cl 11, 12, 13, 14, 15 and 17
  • NMT- vector and DU-145-Vec clones were isolated and maintained as independent cell lines in complete media containing 10 ⁇ g/ml of G418.
  • Northern blotting assays Total cellular RNA was isolated by the guanidinium/phenol extraction method and Northern blotting was performed as described (Su et al., 1997, Proc. Natl. Acad. Sci.
  • Luciferase assays 10 ⁇ l of cell lysate were mixed with 40 ⁇ l of Luciferase Assay substrate (Promega).
  • ⁇ - gals assays 10 ⁇ l of the cell lysate were mixed with 100 ⁇ l of diluted Galecton-Plus with 150 ⁇ l of Accelerator (Tropix). Promoter analysis data were collected a minimum of three times using triplicate samples for each experimental point and the data was standardized with the ⁇ -gal data.
  • rat PSGen 13 Cloning a full length rat PSGen 13 and a HuPSGen 13 cDNA.
  • An original rat PSGen 13 EST was identified using RSDD and reverse Northern hybridization as a gene displaying elevated expression in El 1 versus El 1-NMT cells (Kang et al, 1998, Proc. Natl. Acad. Sci. U.S.A. 95:13788-13793).
  • a full length open reading frame (ORF) of rat PSGen 13 was cloned using the complete open reading frame (C-ORF) approach with gene specific primers and electronic data mining based on the EST sequence.
  • Primers used for C-ORF were PSGenl3-R2 (TCG CTT CTC ACT TTG ACG GAG TGT CAA G) (SEQ ID NO: 7) and PSGenl3-R2 Nested (TGT CAA GTG TGG CAG AGA CTA AGA ATG G) (SEQ ID NO: 8).
  • full length rPSGen 13 and HuPSGen 13 cDNA clones were identified by sequence comparison of the rat PSGen 13 EST sequence with GenBank by BLAST. Selected clones (ATCC #2005777 from rat PSGen 13 and ATCC #2525262 for HuPSGen 13) were procured (Research Genetics) and sequenced.
  • PSGen 13 cDNA consists of 780 bp excluding the poly(A) tail.
  • a poly(A) signal (AATAAA) is located at position 763 (FIGURE 1; SEQ ID NO: 1).
  • the rPSGen 13 cDNA encodes aprotein with predicted 81 amino acids (FIGURE 1 ; SEQ ID NO:2) of calculated molecular weight of 9 kDa with a pi of 5.52.
  • Protein sequence analysis did not indicate hydrophobic patches for membrane spanning regions or signal peptide sequences characteristic of secretory proteins. Motif and pattern analysis also failed to identify sequence homologies with previously reported genes, information that is useful in providing potential insights into the biological function and or mode of action of rat PSGen 13. Based on this observation, rPSGen 13 appears to encode a novel class of proteins.
  • HuPSGen 13 was electronically cloned by analyzing sequences reported in the GenBank data base (FIGURE 2; SEQ ID NO: 3).
  • HuPSGen 13 is 75% identical to rPSGen 13 at the nucleotide level, but 94% identical to rPSGen 13 at the protein level (79/81 residues) (FIGURES 3 and 4).
  • D at 4, K at 38 and I at 77 are conserved substitutions of rPSGen 13 (E at 4, R at 38 and V at 77, respectively), which suggests strong conservation in functionality.
  • sequence identity of the HuPSGen 13 with rPSGen 13 protein coding sequence is 87% at the nucleotide level.
  • HuPSGene is an orthologue of rPSGen 13.
  • the cloned HuPSGen 13 cDNA consists of 835 bp excluding the poly(A) tail and a canonical poly(A) signal was observed at 814 bp. Although an in-frame stop codon was not present, the ORF of HuPSGen 13 starts at the first ATG (197 bp) and runs through 442 bp.
  • HuPSGen 13 encodes 81 amino acids of calculated Mol.
  • rPSGen 13 Suppresses Anchorage Independent Growth in Ell- NMT and DU-145 Cells. rPSGen 13 was identified using RSDD as a gene displaying elevated expression in El 1 cells versus El 1 -NMT cells (Kang et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:13788-13793).
  • rPSGen 13 expression was lower in progressed El 1-NMT, CREF x El 1-NMT Rl and R2, El 1 x El 1-NMT A6TD, El 1 x El 1-NMT ⁇ a, El 1-Ras R12 and El 1-HPV E6/E7 cells.
  • NMT-PSG13 cl number Analysis of the random NMT-PSGen 13 clones, designated as NMT-PSG13 cl number, indicated specific clones displaying no significant reduction in anchorage independence, i.e., NMT-PSG13 cl 3 and 5, and clones displaying reduced agar cloning efficiencies similar to El 1 cells, i.e., NMT-PSG13 cl 7 and 12.
  • NMT-PSG13 cl 7 and 12 a number of clones were also identified that displayed a significant reduction in anchorage independence versus El 1-NMT cells, but less growth suppression than NMT-PSG13 cl 7 and 12, including NMT-PSG13 cl 6, 8, 9, 10, and 11.
  • rPSGen 13 was transfected into DU-145 human prostate cancer cells and random clones were isolated. The clones were then evaluated for anchorage independent growth (FIGURE 8). Several clones were identified, including DU-PEG13 cl 11, 12 and 14, that displayed a significant reduction in anchorage independent growth in comparison with DU-145 cells. Additional clones, including a vector transfected clone and DU-PEG13 cl 13, 15 and 17 displayed similar cloning efficiencies in agar as untransfected DU-145 cells. These results demonstrate that rat PSGen 13 can also suppress the transformed phenotype in human cancer cells, thereby indicating a more general inhibitory capacity of this gene product in tumor cells.
  • Rat PSGen 13 Inhibits Transcriptional Activity in El 1-NMT Cells.
  • El 1-NMT cells display elevated transcription of the PEG-3 and VEGF genes as compared to El 1 cells (Su et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96:15115-15120). These changes in gene expression are believed to be important determinants of the aggressive progressed cancer phenotype of El 1-NMT versus El 1 cells (Id.).
  • rPSGen 13 The RSDD approach has successfully identified a novel gene, rPSGen 13, that can functionally regulate cancer progression in both rodent and human tumor cells.
  • Evidence is presented that forced expression of rPSGen 13 in rodent tumor cells displaying an aggressive cancer phenotype, El 1-NMT, resulted in a suppression of the cancer phenotype as monitored by a reduction in anchorage independent growth.
  • rPSGen 13 when overexpressed in a human prostate cancer cell line (DU- 145), rPSGen 13 also induced an inhibition in anchorage independence.
  • the mechanism by which PSGen 13 induces its cancer growth suppression properties is not known.
  • forced expression of rPSGen 13 directly correlated with a suppression in the transcriptional activities of two significant cancer progression regulating genes, PEG-3 and VEGF.
  • HuPSGen 13 Based on sequence homology, a human PSGen 13 gene, HuPSGen 13, has been isolated. This gene is highly homologous to the rat PSGen 13 gene, 75% and 94% on a nucleotide and protein level, respectively. As shown in the following section, the HuPSGen 13 also has been demonstrated to suppress the transformed phenotype. 7. EXAMPLE: HUMAN PSGEN 13 SUPPRESSES THE
  • HuPSGen 13 suppressed the transformed phenotype of transformed rat embryo cells.
  • Both CREF cells and CREF cells transformed by Haras (“CREF-ras" cells) were transfected with either empty vector or vector containing HuPSGen 13.
  • the vector also contained a hygromycin resistance gene. Transfectants were selected for hygromycin resistance and colony formation in monolayer culture was assessed. The results are shown in FIGURE 11, which depicts the results of triplicate plates, + S.D. This data demonstrates that HuPSGen 13 exerted a selective inhibitory effect on colony formation in the CREF-ray transformed rat embryo fibroblast cells.
  • HuPSGen 13 suppressed the transformed phenotype of human breast cancer cells.
  • Cells of the human breast carcinoma cell line MCF-7 were transfected with HuPSGen 13 contained in a vector that further comprised a hygromycin resistance gene. Transfectants were then selected in hygromycin and isolated clones were evaluated for anchorage independent growth in agar.
  • HuPSGen 13 transfected cell lines exhibited a cloning efficiency similar to untransfected MCF-7 cells (clones 4 and 5), the cloning efficiency was decreased substantially in human PSGen 13 transfected MCF-7 clones 6, 7, 12, 13 and 15.

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Abstract

La présente invention concerne des acides nucléiques isolés codant des protéines du gène 13 (PS Gen 13) à progression supprimée, des vecteurs contenant ces acides nucléiques, des protéines de PSGen 13 isolées, ainsi que des méthodes d'utilisation de ces molécules pour empêcher la croissance de cellules cancéreuses et/ou de nouveaux vaisseaux sanguins et soigner en conséquence les patients cancéreux. Ces méthodes reposent, au moins en partie, sur la caractérisation des ADNc complets codant le PSGen 13 humain et murin, et sur la découverte selon laquelle les niveaux d'expression élevés de PSGen 13 peuvent supprimer le phénotype transformé et inhiber les activités des promoteurs associées à l'évolution du cancer et à l'angiogenèse. Divers modes de réalisation utilisent une méthode permettant d'inhiber la croissance des cellules cancéreuses. Cette méthode consiste à mettre en contact les cellules cancéreuses avec une dose suffisante d'acide nucléique codant une protéine PSGen 13, une protéine PSGen 13 ou un activateur de PSGen 13, pour inhiber la croissance des cellules cancéreuses. Par ailleurs, l'invention concerne une méthode de traitement du cancer chez un sujet, consistant à mettre en contact les cellules du sujet avec une dose suffisante d'un acide nucléique codant une protéine PSGen 13 pour amener la cellule à exprimer la protéine PSGen 13 et traiter ainsi le cancer chez le sujet.
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